Clinical disability in multiple sclerosis (MS) is driven by immune cell infiltration into the CNS parenchyma. Understanding the mechanisms by which CNS barrier cells regulate immune cell activation and entry during this infiltrative process may identify translational strategies against MS and other autoimmune CNS diseases. During CNS inflammation, immune cells traffic from the blood through a two-barrier structure termed the neurovascular unit. While many have focused on entry through the first barrier, a specialized endothelial wall known as the blood-brain barrier, less is known about how immune cells interact with the second barrier, a layer of astrocyte end feet termed the glia limitans. In MS lesions, immune cells circulate in the space between the blood-brain barrier and the glia limitans, a compartment called the perivascular space. Here, astrocytes may interact with infiltrating immune cells to regulate their function. Using gene expression analysis, we identified immune cell receptors upregulated by astrocytes when treated with interleukin-1?, a cytokine in MS lesions. One of our strongest candidates, Junctional Adhesion Molecule-A (JAM-A), is an immune cell receptor that regulates pro-inflammatory pathways in other tissues. My pilot data reveals that astrocytic JAM-A Binds T cells and alters protease and cytokine levels in patterns known to exacerbate an animal model of MS. Further pilot data show that astrocytic JAM-A deletion blocks immune cell penetration of the glia limitans and protects against clinical disability in this model. I hypothesize that astrocytic JAM-A drives CNS entry of immune cells, inflammatory lesion formation and clinical disability. In Aim 1, I will test how astrocytic JAM-A signaling to T cells in co-culture modifies gene expression networks of both cell types using RNA sequencing, and then I will determine how JAM-A and downstream candidates regulate effector pathways relevant to autoimmune attack. In Aim 2, using transgenic and pharmacologic tools, I will establish how astrocytic JAM-A controls lesion formation in two animal models of CNS inflammation. Translational strategies blocking astrocytic JAM-A and effectors will then be tested against clinical disability in an animal model of MS. In Aim 3, I will measure how T cells interact with the glia limitans using live two-photon imaging of inflammatory spinal cord lesions. I will then test how astrocytic JAM-A and effectors regulate these interactions. Goals of this K08 award are to advance my expertise in neuroimmunology and glial biology under the mentorships of Gareth John, PhD., Peter Calabresi, MD. and Dimitrios Davalos, PhD., to acquire technical skills using immune and glial cell co-cultures and in vivo multiphoton imaging, and to establish my own research group focused on the role of astrocyte-immune cell signaling during autoimmune attack. This K08 plan investigates a novel contact-mediated interaction between reactive astrocytes and infiltrating immune cells within an understudied immunomodulatory compartment of the CNS and may identify novel therapeutic strategies against CNS autoimmune disease.